Super-Resolution Dissection of Coordinated Events during Malaria Parasite Invasion of the Human Erythrocyte

Erythrocyte invasion by the merozoite is an obligatory stage in Plasmodium parasite infection and essential to malaria disease progression. Attempts to study this process have been hindered by the poor invasion synchrony of merozoites from the only in vitro culture-adapted human malaria parasite, Plasmodium falciparum. Using fluorescence, three-dimensional structured illumination, and immunoelectron microscopy of filtered merozoites, we analyze cellular and molecular events underlying each discrete step of invasion. Monitoring the dynamics of these events revealed that commitment to the process is mediated through merozoite attachment to the erythrocyte, triggering all subsequent invasion events, which then proceed without obvious checkpoints. Instead, coordination of the invasion process involves formation of the merozoite-erythrocyte tight junction, which acts as a nexus for rhoptry secretion, surface-protein shedding, and actomyosin motor activation. The ability to break down each molecular step allows us to propose a comprehensive model for the molecular basis of parasite invasion. © 2011 Elsevier Inc

Plasmodium falciparum Merozoite Invasion Is Inhibited by Antibodies that Target the PfRh2a and b Binding Domains

Plasmodium falciparum, the causative agent of the most severe form of malaria in humans invades erythrocytes using multiple ligand-receptor interactions. The P. falciparum reticulocyte binding-like homologue proteins (PfRh or PfRBL) are important for entry of the invasive merozoite form of the parasite into red blood cells. We have analysed two members of this protein family, PfRh2a and PfRh2b, and show they undergo a complex series of proteolytic cleavage events before and during merozoite invasion. We show that PfRh2a undergoes a cleavage event in the transmembrane region during invasion consistent with activity of the membrane associated PfROM4 protease that would result in release of the ectodomain into the supernatant. We also show that PfRh2a and PfRh2b bind to red blood cells and have defined the erythrocyte-binding domain to a 15 kDa region at the N-terminus of each protein. Antibodies to this receptor-binding region block merozoite invasion demonstrating the important function of this domain. This region of PfRh2a and PfRh2b has potential in a combination vaccine with other erythrocyte binding ligands for induction of antibodies that would block a broad range of invasion pathways for P. falciparum into human erythrocytes

An EGF-like Protein Forms a Complex with PfRh5 and Is Required for Invasion of Human Erythrocytes by Plasmodium falciparum

Invasion of erythrocytes by Plasmodium falciparum involves a complex cascade of protein-protein interactions between parasite ligands and host receptors. The reticulocyte binding-like homologue (PfRh) protein family is involved in binding to and initiating entry of the invasive merozoite into erythrocytes. An important member of this family is PfRh5. Using ion-exchange chromatography, immunoprecipitation and mass spectroscopy, we have identified a novel cysteine-rich protein we have called P. falciparum Rh5 interacting protein (PfRipr) (PFC1045c), which forms a complex with PfRh5 in merozoites. Mature PfRipr has a molecular weight of 123 kDa with 10 epidermal growth factor-like domains and 87 cysteine residues distributed along the protein. In mature schizont stages this protein is processed into two polypeptides that associate and form a complex with PfRh5. The PfRipr protein localises to the apical end of the merozoites in micronemes whilst PfRh5 is contained within rhoptries and both are released during invasion when they form a complex that is shed into the culture supernatant. Antibodies to PfRipr1 potently inhibit merozoite attachment and invasion into human red blood cells consistent with this complex playing an essential role in this process

Heparin assisted assembly of somatostatin amyloid nanofibrils results in disordered precipitates by hindrance of protofilaments interactions

Amyloid nanofibrils are β-sheet rich protein or peptide assemblies that have pathological roles in over 20 neurodegenerative diseases, but also can have essential physiological roles. This wide variety of functions is likely to be due to subtle differences in amyloid structure and assembly mechanisms. Glycosaminoglycans (GAGs), like heparin, are frequently used in vitro to increase the kinetics of assembly of amyloid fibrils. However, little is known about the effects of adding large polymeric sugars on assembly mechanisms and amyloid nanostructures. Here, we provide insights into the kinetics, assembly mechanisms and structural effects of heparin on the self-assembly of a functional-amyloid forming neuropeptide hormone, somatostatin-14. We show that pure somatostatin-14 self-assembles into amyloid fibrils via the formation of antiparallel β-sheet networks, in a typical amyloid aggregation process. These fibrils then laterally assemble into ordered liquid crystalline structures through the generation of further parallel β-sheet networks. If heparin molecules are present, they intercalate between the peptide assemblies during the initial stages of aggregation. This intercalation screens electrostatic repulsions hindering the lateral association of protofilaments, preventing liquid crystal formation and resulting in the rapid formation of disordered micron scale precipitates. Our results show that aggregation promotors like heparin can have large effects not just on the kinetics of aggregation but also on assembly mechanisms, and the architecture of amyloid assemblies. Thus highlighting the dangers of using such polymeric sugars in fundamental studies of amyloid aggregation, especially when drawing conclusions on structure-function relationships or when investigating amyloid-based nanostructures as bionanomaterials

Malaria parasite signal peptide peptidase is an ER-resident protease required for growth but not for invasion

The establishment of parasite infection within the human erythrocyte is an essential stage in the development of malaria disease. As such, significant interest has focused on the mechanics that underpin invasion and on characterization of parasite molecules involved. Previous evidence has implicated a presenilin-like signal peptide peptidase (SPP) from the most virulent human malaria parasite, Plasmodium falciparum, in the process of invasion where it has been proposed to function in the cleavage of the erythrocyte cytoskeletal protein Band 3. The role of a traditionally endoplasmic reticulum (ER) protease in the process of red blood cell invasion is unexpected. Here, using a combination of molecular, cellular and chemical approaches we provide evidence that PfSPP is, instead, a bona fide ER-resident peptidase that remains intracellular throughout the invasion process. Furthermore, SPP-specific drug inhibition has no effect on erythrocyte invasion whilst having low micromolar potency against intra-erythrocytic development. Contrary to previous reports, these results show that PfSPP plays no role in erythrocyte invasion. Nonetheless, PfSPP clearly represents a potential chemotherapeutic target to block parasite growth, supporting ongoing efforts to develop antimalarial-targeting protein maturation and trafficking during intra-erythrocytic development.Danushka S. Marapana, Danny W. Wilson, Elizabeth S. Zuccala, Chaitali D. Dekiwadia, James G. Beeson, Stuart A. Ralph and Jake Bau

Evaluation of cytotoxic and apoptotic activities of Clinacanthus nutans (Burm. f.) Lindau leaves against D24 human melanoma cells

The leaves of Clinacanthus nutans have been increasingly used as an adjuvant treatment for cancers. Infusion of the dried leaves in water is the traditional and preferred method of preparation by cancer patients for consumption. Traditionally, this medicinal plant is brewed in boiling water before consumption as a tea but little is known about the effect of preparation temperature for optimal anticancer activity. Therefore, the present study assessed the effect of preparation temperature (hot or cold) of crude aqueous extracts of C. nutans leaves on the cytotoxicity of D24 melanoma cells using a colorimetric WST-8 assay. Apoptotic cell death was detected by a cytofluorometric double staining method. Biochemical and morphological changes in the treated cells were examined by fluorescence and transmission electron microscopy techniques, respectively, to further verify the type of cell death. The cold aqueous extract was the most cytotoxic against D24 melanoma cells with an EC50 of 163 µg/mL at 72 h. The extract also predominantly induced apoptosis in these cells according to cytofluorimteric, confocal and transmission electron microscopic analyses. This study provides important information on the preparation of C. nutans and its effectiveness as an anticancer concoction for regular consumption

Electrospun nanodiamond-silk fibroin membranes: a multifunctional platform for biosensing and wound-healing applications

Next generation wound care technology capable of diagnosing wound parameters, promoting healthy cell growth, and reducing pathogenic infections noninvasively would provide patients with an improved standard of care and accelerated wound repair. Temperature is one of the indicating biomarkers specific to chronic wounds. This work reports a hybrid, multifunctional optical material platform-nanodiamond (ND)-silk membranes as biopolymer dressings capable of temperature sensing and promoting wound healing. The hybrid structure was fabricated through electrospinning, and 3D submicron fibrous membranes with high porosity were formed. Silk fibers are capable of compensating for the lack of an extracellular matrix at the wound site, supporting the wound-healing process. Negatively charged nitrogen vacancy (NV-) color centers in NDs exhibit optically detected magnetic resonance (ODMR) and act as nanoscale thermometers. This can be exploited to sense temperature variations associated with the presence of infection or inflammation in a wound, without physically removing the dressing. Our results show that the presence of NDs in the hybrid ND-silk membranes improves the thermal stability of silk fibers. NV- color centers in NDs embedded in silk fibers exhibit well-retained fluorescence and ODMR. Using the NV- centers as fluorescent nanoscale thermometers, we achieved temperature sensing in 25-50 °C, including the biologically relevant temperature window, for cell-grown ND-silk membranes. An enhancement (∼1.5× on average) in the temperature sensitivity of the NV- centers was observed for the hybrid materials. The hybrid membranes were further tested in vivo in a murine wound-healing model and demonstrated biocompatibility and equivalent wound closure rates as the control wounds. Additionally, the hybrid ND-silk membranes exhibited selective antifouling and biocidal propensity toward Gram-negative Pseudomonas aeruginosa and Escherichia coli, while no effect was observed on Gram-positive Staphylococcus aureus.Asma Khalid, Dongbi Bai, Amanda N. Abraham, Amit Jadhav, Denver Linklater, Alex Matusica ... et al

PC 12 pheochromocytoma cell response to super high frequency terahertz radiation from synchrotron source

High frequency (HF) electromagnetic fields (EMFs) have been widely used in many wireless communication devices, yet within the terahertz (THz) range, their effects on biological systems are poorly understood. In this study, electromagnetic radiation in the range of 0.319.5 × 10 12 Hz, generated using a synchrotron light source, was used to investigate the response of PC 12 neuron-like pheochromocytoma cells to THz irradiation. The PC 12 cells remained viable and physiologically healthy, as confirmed by a panel of biological assays; however, exposure to THz radiation for 10 min at 25.2 ± 0.4 ◦ C was sufficient to induce a temporary increase in their cell membrane permeability. High-resolution transmission electron microscopy (TEM) confirmed cell membrane permeabilization via visualisation of the translocation of silica nanospheres (d = 23.5 ± 0.2 nm) and their clusters (d = 63 nm) into the PC 12 cells. Analysis of scanning electron microscopy (SEM) micrographs revealed the formation of atypically large (up to 1 µm) blebs on the surface of PC 12 cells when exposed to THz radiation. Long-term analysis showed no substantial differences in metabolic activity between the PC 12 cells exposed to THz radiation and untreated cells; however, a higher population of the THz-treated PC 12 cells responded to the nerve growth factor (NGF) by extending longer neurites (up to 020 µm) compared to the untreated PC12 cells (up to 20 µm). These findings present implications for the development of nanoparticle-mediated drug delivery and gene therapy strategies since THz irradiation can promote nanoparticle uptake by cells without causing apoptosis, necrosis or physiological damage, as well as provide a deeper fundamental insight into the biological effects of environmental exposure of cells to electromagnetic radiation of super high frequencies